New analytical methods using fluorescence detection are becoming increasingly quantitative and require easy-to-use material standards for fluorometer qualification and method validation. NIST is responding to this need by developing and producing such standards. Reported here is the first step in this process, which is to qualify a research-grade fluorescence spectrometer for measuring true fluorescence spectra of reference material candidates. "True" spectra are defined here as those with fluorescence intensity, either relative or absolute as required, and wavelength both being reported with high accuracy and known precision, after wavelength has been calibrated and corrections for excitation intensity and detection system response have been applied. The uncertainties determined in relative and absolute intensity-corrected fluorescence spectra using both calibrated source (CS)- and calibrated detector (CD)-based methods were compared. The CS-based method gave uncertainties, typically about +/-5% for relative spectral correction, that were about half that of the CD-based method for determining both relative and absolute spectral correction factors. Absolute spectral correction factors can be determined using either method without knowing the optical geometry of the instrument. The absolute spectral correction factors were found to have much larger uncertainties than the corresponding relative correction factors with uncertainties for the CS-based method of +/-10% to +/-15% being typical and +/-20% or more not being uncommon, particularly for excitation and emission wavelengths below 400 nm. Uncertainties arising from detection system nonlinearity and instrument polarization ratios were also explored.
Chromophore-based fluorescence standards for the characterization of photoluminescence measuring systems and the determination of relevant fluorometric quantities are classified according to their scope and area of application. General and type-specific requirements for suitable standards are derived for each class of standards. Metrological requirements linked to the realization of comparable measurements are addressed and recommendations on selecting, using, and developing fluorescence standards are given.
Aimed at improving quality assurance and quantitation for modern fluorescence techniques, ASTM International (ASTM) is about to release a Standard Guide for Fluorescence, reviewed here. The guide's main focus is on steady state fluorometry, for which available standards and instrument characterization procedures are discussed along with their purpose, suitability, and general instructions for use. These include the most relevant instrument properties needing qualification, such as linearity and spectral responsivity of the detection system, spectral irradiance reaching the sample, wavelength accuracy, sensitivity or limit of detection for an analyte, and day-to-day performance verification. With proper consideration of method-inherent requirements and limitations, many of these procedures and standards can be adapted to other fluorescence techniques. In addition, procedures for the determination of other relevant fluorometric quantities including fluorescence quantum yields and fluorescence lifetimes are briefly introduced. The guide is a clear and concise reference geared for users of fluorescence instrumentation at all levels of experience and is intended to aid in the ongoing standardization of fluorescence measurements.
Procedures for the characterization of photoluminescence measuring systems are discussed, focusing on spectrofluorometers and fit-for-purpose methods including suitable standards. The aim here is to increase the awareness for the importance of a reliable instrument characterization and to improve the reliability and comparability of measurements of photoluminescence.
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The NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing. It must therefore embody the quality and characteristics of a typical biopharmaceutical product and be available long-term in a stable format with consistent product quality attributes. A stratified sampling and analysis plan using a series of qualified analytical and biophysical methods is described that assures RM 8671 meets these criteria. Results for the first three lots of RM 8671 highlight the consistency of material attributes with respect to size, charge, and identity. RM 8671 was verified to be homogeneous both within and between vialing lots, demonstrating the robustness of the lifecycle management plan. It was analyzed in concert with the in-house primary sample 8670 (PS 8670) to provide a historical link to this seminal material. RM 8671 was verified to be fit for its intended purpose as a technology innovation tool, external system suitability control, and cross-industry harmonization platform.
Graphical abstractThe NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing.
Electronic supplementary materialThe online version of this article (10.1007/s00216-017-0800-1) contains supplementary material, which is available to authorized users.
Standard Reference Material s (SRM s ) 2941 is a cuvette-shaped, uranyl-ion-doped glass, recommended for use for relative spectral correction of emission and day-to-day performance validation of fluorescence spectrometers. Properties of this standard that influence its effective use or contribute to the uncertainty in its certified emission spectrum have been explored here. These properties include its photostability, absorbance, dissolution rate in water, anisotropy, temperature coefficient of fluorescence intensity, and fluorescence lifetimes. The expanded uncertainties in the certified spectrum are about 4% around the peak maximum at 526 nm, using an excitation wavelength of 427 nm. The SRM also exhibits a strong resistance to photodegradation, with no measurable decrease in fluorescence intensity even after 8 h of laser irradiation. Published by Elsevier B.V.
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